PROJECT SUMMARY To understand oligodendrocyte differentiation and myelin formation in the CNS, it is essential to define how intracellular signaling pathways regulate the cytoplasmic and nuclear events that drive myelination. A number of signaling pathways have been implicated in driving CNS myelination. The collaborative project between the Macklin and Wood laboratories was initiated to define the function specifically of mTOR signaling in oligodendrocyte and myelin biology. Over the past several years, our laboratories have contributed significant findings on the function of mTOR and its associated complexes through this successful collaboration. The current application addresses a fundamental gap in understanding the mechanisms by which mTOR regulates oligodendrocyte differentiation and myelination through regulating specific downstream targets. Our earlier studies demonstrated that oligodendrocyte loss of mTOR or raptor, the mTORC1-associated protein, results in deficits in oligodendrocyte differentiation and initiation of myelination in the spinal cord. Moreover, we found that these mice have reduced myelin thickness that was sustained in the adult spinal cord. A significant strength of the studies is that we have also begun to define distinct mTOR-dependent and ?independent pathways that regulate developmental myelination in the brain versus spinal cord. The goal of the current studies is to address the fundamental questions of 1) what are the mTOR-dependent mechanisms that regulate the conversion of oligodendrocyte progenitors to differentiating oligodendrocytes through regulating transcriptional machinery, 2) how does mTOR regulate specific cytoskeletal changes necessary for initiation of myelination and myelin wrapping, and 3) how does the crosstalk between mTORC2 and integrin-linked kinase (ILK) pathways regulate myelination in the corpus callosum? We will address these questions in both rodent and zebrafish models by testing the following hypotheses: 1) mTOR promotes oligodendrocyte differentiation by suppressing bone morphogenetic pathway (BMP) signaling and down-regulating transcriptional inhibitors, 2) mTOR promotes initiation of myelination through regulating specific cytoskeletal targets during both process extension and axon wrapping, and 3) rictor regulates myelination in the corpus callosum through promoting Akt473 phosphorylation in coordination with both mTOR and ILK.